In our prior studies utilizing myeloma specific antigen (idiotype) and multiple myeloma (MM) cell-based vaccination, we observed induction of antigen-specific immune responses; however, clinical responses were not seen. To achieve clinically meaningful immune response, in the previous funding period, we further investigated immune-competence in MM. We evaluated development of immune response to Hepatitis B vaccination in patients with MM and monoclonal gammopathy of undetermined significance (MGUS) and observed that immune function is significantly impaired in MM and interestingly also in patients with MGUS. Our further investigations have identified both dysfunctional T regulatory cells that affect immune homeostasis in myeloma, and up-regulated Th17 cells that affects both myeloma cell growth and survival as well as suppress Th1 immune responses. Moreover these cells produce cytokines (IL-17, IL-21, IL-22, IL-23 and IL- 27) with significant immunosuppressive activity. A significant body of information has emerged supporting a critical role for the bone marrow (BM) microenvironment in supporting not only MM cell growth, and survival, but also in inducing the immune dysfunction. Based on the information that interleukin-6 (IL-6), transforming growth factor-beta (TGF-), and IL-1 are elevated in MM and may play an important role in T cell function we hypothesize that conditions generated in BM microenvironment by interaction between MM cells and bone marrow stromal cells (BMSC) modulate the immune responses to support tumor progression in MGUS and in MM and targeting these conditions may allow us to improve immune responses and develop immunotherapeutic strategies. Towards this overall objective we will evaluate the role of regulatory T cells (Treg) and their imbalance with TH17 cells in the promotion of immune dysfunction and tumor growth in MM (Specific Aim 1). In this objective we will first investigate both qualitative and quantitative aberrations and molecular determinants of regulatory T cell dysfunction and its interplay with Th17 cells in the BM microenvironment and peripheral circulation in MM and MGUS. We will utilize paired samples from patients' with MGUS progressing to myeloma to understand the change in immune make up from MGUS progression to MM. Additionally, we will evaluate the direct and indirect effects of pro-inflammatory cytokines produced by Treg and Th17 cells on MM cell growth and survival and immune response. We will investigate modulators of immune responses to overcome immune suppressive effects observed in MM to augment effector T cell function (Specific Aim 2). We will characterize the anti-MM effector T cell responses in peripheral blood and bone marrows of MGUS and MM patients compared with normal individuals against MM-related antigens Xbp-1, OFD-1 and Sox-2 and then evaluate modulators of immune function (anti- IL-17, anti-IL-6 & Revlimid) alone and in combination to improve T effector cell-functions in MM. As we define the mediators of immune suppression in MM and investigate agents able to overcome the suppressive effects, we will develop antigen specific peptide-based vaccination strategy in combination with modulators of immune function. (Specific Aim 3). We have analyzed our large clinically annotated gene expression profile, alternate splicing and copy number alterations data from myeloma patients and identified and validated clinically critical genes. We will now evaluate immunologically relevant peptides targeting these genes for CTL response. Finally we will combine the immune modulators and peptide vaccination to generate robust immune response. The proposed studies will identify the mechanism of immune suppression in myeloma, develop methods to improve immune function and develop peptide-based vaccination strategies to preclinical rational for their clinical application.